Purification of tobacco-derived protein compositions

ABSTRACT

The disclosure describes methods for the purification of protein-enriched extracts to provide concentrates and isolates and methods for incorporation of such materials into products. The purification methods are adapted for removal of nicotine and may have other benefits, e.g., lightening the color of the protein-enriched extracts. The methods generally include treatment with peracetic acid or hydrogen peroxide and filtrations. A protein composition in the form of a concentrate or isolate is provided, the protein composition including RuBisCO, F2 fraction proteins, or combination thereof extracted from a plant of the  Nicotiana  species, wherein the protein composition is characterized by relatively low nicotine content.

FIELD OF THE INVENTION

The present invention relates primarily to products made or derived fromtobacco, or that otherwise incorporate tobacco or components of tobacco,and are intended for human consumption. Of particular interest areingredients or components obtained or derived from plants or portions ofplants from the Nicotiana species.

BACKGROUND OF THE INVENTION

Many uses of tobacco have been proposed. For example, tobacco has beensmoked in pipes, and tobacco has been used for smoking in cigarettes andcigars. See, for example, Tobacco Production, Chemistry and Technology,Davis et al. (Eds.) p. 346 (1999). In recent years, there have beenproposed various ways of providing many of the sensations of smoking,but without delivering considerable quantities of incomplete combustionand pyrolysis products that result from burning tobacco. See, forexample, the background art set forth in U.S. Pat. No. 7,503,330 toBorschke et al. and U.S. Pat. No. 7,726,320 to Robinson et al., and U.S.patent application Ser. No. 13/840,264 to Novak, III et al., filed Mar.15, 2013 and Ser. No. 13/647,670 to Gerardi, filed Oct. 9, 2012. Tobaccoalso has been enjoyed in so-called smokeless forms. See, for example,the background art set forth in US Pat. Pub. 2012/0272976 to Byrd et al.In addition, various materials derived from tobacco have been proposedto have uses in certain industrial applications. See, for example, U.S.Pat. No. 2,098,836 to Ressler; U.S. Pat. No. 2,232,662 to Hockenyos;U.S. Pat. No. 4,347,324 to Wildman et al. and U.S. Pat. No. 4,289,147 toWildman et al., and US Pat. Pub. Nos. 2011/01287681 to DeVall and2012/0260929 to Coleman et al.

Methods of extracting proteins from tobacco and tobacco components havebeen proposed in U.S. Pat. No. 9,301,544 to Mua et al., which isincorporated herein by reference. Certain methods for purifying suchproteins and for compositions incorporating such proteins have beendescribed in U.S. Pat No. 9,175,052 to Gerardi et al. and US Pat. App.Publ. No. 2016/0029663 to Gerardi et al., respectively. It would bedesirable to provide further methods for purifying tobacco-derivedproteins, e.g., for inclusion in tobacco formulations useful in themanufacture of smoking articles and/or smokeless tobacco products, aswell as for incorporation into other products such as nutritionalsupplements.

SUMMARY OF THE INVENTION

The present invention provides materials derived from plants,particularly from plants of the Nicotiana species. In preferredembodiments, the materials are provided in what can be considered to besubstantially purified form. The invention also provides methods forpurifying components from plants, e.g., plants of the Nicotiana species,and methods for further processing those components. In particular, theinvention provides protein concentrates, isolates, and other forms ofprotein-containing products derived from tobacco material (andparticularly, such concentrates, isolates, and other forms with reducednicotine content). The invention further provides methods for obtainingsuch protein concentrates, isolates, and other forms ofprotein-containing products, and methods for incorporation of suchprotein concentrates, isolates, and other forms of protein-containingproducts into various types of compositions.

In one aspect, the present disclosure provides a method for reducing thenicotine content in a plant-derived protein-enriched material, themethod comprising: a) receiving a plant-derived, protein-enrichedmaterial comprising RuBisCO, F2 fraction proteins, or a combinationthereof, wherein the plant-derived, protein-enriched material furthercomprises undesirable nicotine; b) treating the plant-derived,protein-enriched material with peracetic acid to give a peraceticacid-treated mixture; c) washing the peracetic acid-treated mixture withwater on a filter, wherein a solid treated protein-containing materialis retained on the filter; d) solubilizing the solid treatedprotein-containing material to give a solution and adjusting the pH ofthe solution to a basic pH to give a basic solution; and e) processingthe basic solution on a filter to afford a retentate comprising aprotein concentrate or isolate having a reduced amount of nicotine ascompared with the plant-derived, protein-enriched material. In someembodiments, the plant-derived, protein-enriched material comprisesmaterial from a plant of the Nicotiana species.

Various parameters can be adjusted with respect to each step of thismethod. In the context of the foregoing method, in some embodiments,treating step b) is conducted at a pH of about 3 to about 5. In someembodiments, the treating step b) is conducted at a temperature of about25° C. to about 42° C. Treating step b), in certain embodiments,comprises treating the plant-derived, protein-enriched material withperacetic acid in the form of an aqueous solution comprising at leastabout 3% by weight peracetic acid. Washing step c) can, in someembodiments, comprise washing the peracetic acid-treated mixture withwater on a 1.4 μm pore filter. Washing step c) is advantageously doneusing acidic water, e.g., water having a pH of less than about 5. Insome embodiments, step d) comprises resolubilizing the solid treatedprotein-containing material in an aqueous buffer. The aqueous buffer canvary; in particular embodiments, the aqueous buffer comprises sodiumbicarbonate and potassium carbonate. In some embodiments, step e)comprises processing the basic solution on a 10 nm filter. The disclosedmethod may, in certain embodiments, further comprise spray drying theprotein concentrate or isolate.

Advantageously, the resulting protein concentrate or isolate typicallyexhibits decreased nicotine content with respect to the as-receivedplant-derived, protein-enriched material. For example, the proteinconcentrate or isolate in certain embodiments exhibits a nicotinecontent of about 5 ppm or less or about 2 ppm or less by dry weight. Insome embodiments, the protein concentrate or isolate exhibits a greateroverall concentration by dry weight of protein (RuBisCO, F2 fractionproteins, or a combination thereof) than in the as-received material.For example, in some embodiments, the concentration can be about 80% orgreater by dry weight, e.g., about 80% to about 90% by dry weight.Further, the protein concentrate or isolate in some embodiments ischaracterized as having better sensory characteristics and can, in someembodiments, be substantially odorless, substantially colorless, and/orsubstantially tasteless.

In another aspect, the disclosure provides a method for reducing thenicotine content in a plant-derived protein-enriched material, themethod comprising: a) receiving a plant-derived, protein-enrichedmaterial comprising RuBisCO, F2 fraction proteins, or a combinationthereof, wherein the plant-derived, protein-enriched material furthercomprises undesirable nicotine; b) treating the plant-derived,protein-enriched material with a basic solution to give a base-treatedmixture; c) mixing the base-treated mixture with hydrogen peroxide togive a hydrogen-peroxide-treated material; and d) processing the basicsolution on a filter to afford a retentate comprising a proteinconcentrate or isolate having a reduced amount of nicotine as comparedwith the plant-derived, protein-enriched material.

Various parameters can be adjusted with respect to each step of thismethod. In the context of the foregoing method, in some embodiments,treating step b) comprises treating the plant-derived, protein-enrichedmaterial with the basic solution to a pH of about 10 or greater. In someembodiments, the method further comprises heating the base-treatedmixture before, during, or both before and during mixing step c). Forexample, the base-treated mixture can be heated at a temperature ofabout 30° C. to about 42° C. In certain embodiments, processing step d)comprises processing the basic solution on a filter with a pore size ofabout 10 nm or a molecular weight cutoff of about 10 kDa. The methodcan, in some embodiments, further comprise spray drying the proteinconcentrate or isolate. Advantageously, the resulting proteinconcentrate or isolate typically exhibits decreased nicotine contentwith respect to the as-received plant-derived, protein-enrichedmaterial. For example, the protein concentrate or isolate in certainembodiments exhibits a nicotine content of about 30 ppm or less by dryweight.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to provide an understanding of embodiments of the invention,reference is made to the appended drawings, which are not necessarilydrawn to scale, and in which reference numerals refer to components ofexemplary embodiments of the invention. The drawings are exemplary only,and should not be construed as limiting the invention.

FIG. 1 is a flowchart of method steps associated with one embodiment ofthe present disclosure;

FIG. 2 is a flowchart of method steps associated with another embodimentof the present disclosure;

FIG. 3 is an exploded perspective view of a smoking article having theform of a cigarette, showing the smokable material, the wrappingmaterial components, and the filter element of the cigarette; and

FIG. 4 is a cross-sectional view of a smokeless tobacco productembodiment, taken across the width of the product, showing an outerpouch filled with a smokeless tobacco composition of the invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention now will be described more fully hereinafter. Thisinvention may, however, be embodied in many different forms and shouldnot be construed as limited to the embodiments set forth herein; rather,these embodiments are provided so that this disclosure will be thoroughand complete, and will fully convey the scope of the invention to thoseskilled in the art. As used in this specification and the claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontext clearly dictates otherwise. Reference to “dry weight percent” or“dry weight basis” refers to weight on the basis of dry ingredients(i.e., all ingredients except water).

Generally, the present invention provides methods for purifyingbiomass-derived protein extracts. In one preferred embodiment, thebiomass-derived protein extract comprises protein derived from a plantof the Nicotiana species. Often, methods of deriving protein fromvarious biomasses results in a protein-enriched material that is notpure protein and may contain various other components extracted from thebiomass. For example, in certain embodiments the crude protein-enrichedmaterial extracted from the biomass can further comprise such substancesas nicotine, lipids, chlorophyll, tannins, minerals, plant structuralcomponents, and phenolic pigments.

Although the present disclosure focuses primarily on proteins extractedfrom tobacco, it is noted that the methods may be generally applicableto crude protein materials extracted from plant materials other thantobacco. The methods described herein are thus intended to be applicableto any plant comprising green leaves and/or any plant believed tocomprise proteins. In some embodiments, the methods are applicable totrees, bushes, grasses, ferns, vines, mosses, algae, and herbs. Forexample, the methods for preparing and purifying a protein-enrichedmaterial according to the present disclosure are in some embodimentsapplicable to such plants as spinach, alfalfa, Swiss chard, kale,chicory, amaranth, barley leaves, mustard greens, clover, carrot leaves,and beet leaves.

According to the present disclosure, a protein-enriched material can befurther processed to provide a purer protein composition, such as acomposition in the form of a concentrate or isolate. By“protein-enriched material” is meant a material (e.g., an extract) thathas been derived from a plant and which contains one or more types ofprotein. The crude protein-enriched materials described herein generallycomprise an amount of undesirable components/impurities such as ash,metal salts, trace metals, alkaloids (e.g., nicotine), precipitates, andother residual materials. Further, the protein-enriched materials mayexhibit undesirable sensory or organoleptic characteristics (e.g., tastecharacteristics, odor, and/or color). By “protein concentrate” as usedherein is meant a material comprising between about 29% and about 89% byweight protein on a dry weight basis. By “protein isolate” as usedherein is meant a material comprising about 90% or more protein byweight on a dry weight basis.

Advantageously according to the invention, methods are provided forpurifying protein-enriched materials to provide concentrates and/orisolates containing minimal amounts of certain undesirablecomponents/impurities. Various methods disclosed herein can providepurified protein isolates comprising about 60% or greater or about 65%or greater by weight protein and low nicotine levels, e.g., about 50 ppmor lower, about 30 ppm or lower, about 20 ppm or lower, about 10 ppm orlower, or about 5 ppm or lower nicotine.

The present disclosure is applicable, in some embodiments, for largescale production, where the term large scale production refers toprocessing large quantities of a biomass on a mass production level. Theterm “biomass” and related terms such as “biomatter” and “plant source”are understood to refer to any portion of a harvested plant that may beprocessed to extract, separate, or isolate components of interesttherefrom. The processing may be carried out in relation to variousplants or portions thereof, such as seeds, flowers, stalks, stems,roots, tubers, leaves, or any further portions of the plant.

In particular embodiments, the methods described herein can beparticularly relevant to the isolation and/or purification of a crudeprotein-containing material (e.g., extract) obtained from a tobaccoplant material or a portion thereof. Exemplary tobacco plant materialsused in accordance with the present disclosure may be of some form of aplant of the Nicotiana species, as described for example, in U.S. Pat.No. 9,254,001 to Byrd et al., which is incorporated by reference herein.Further descriptions of various types of tobaccos, growing practices andharvesting practices are set forth in Tobacco Production, Chemistry andTechnology, Davis et al. (Eds.) (1999), which is incorporated herein byreference. Additional information on types of Nicotiana species suitablefor use in the present invention can be found in U.S. Pat. No. 9,107,453to Dube et al., which is incorporated by reference herein. The plantmaterial may comprise material derived from an entire plant or anyportion of a plant of the Nicotiana species. See, for example, theportions of tobacco plants set forth in U.S. Pat. No. 8,955,523 toColeman, III et al. and U.S. Pat. No. 9,107,453 to Dube et al., whichare incorporated by reference herein.

The plant of the Nicotiana species can be employed in either an immatureor mature form, and can be used in either a green form or a cured form,as described in U.S. Pat. No. 9,107,453 to Dube et al., which isincorporated by reference herein. The tobacco material can be subjectedto various treatment processes such as, refrigeration, freezing, drying(e.g., freeze-drying or spray-drying), irradiation, yellowing, heating,cooking (e.g., roasting, frying or boiling), fermentation, bleaching orotherwise subjected to storage or treatment for later use. In someembodiments, harvested tobacco can be sprayed with a buffer orantioxidant (e.g., a sodium metabisulfite buffer) to prevent the greenplants from browning prior to further treatment as described herein.Other exemplary processing techniques are described, for example, inU.S. Pat. No. 7,946,295 to Brinkley et al. and U.S. Pat. No. 8,955,523to Coleman, III et al., which are incorporated by reference herein. Atleast a portion of the plant of the Nicotiana species can be treatedwith enzymes and/or probiotics before or after harvest, as discussed inUS Pat. Appl. Pub. No. 2013/0269719 to Marshall et al. and U.S. Pat. No.9,485,953 to Moldoveanu, which are incorporated herein by reference.

In particular embodiments, the proteins of the protein-enrichedmaterials to be treated (as well as the proteins comprising theresulting concentrates and isolates described herein) can compriseFraction 1 (“F1”) proteins and/or Fraction 2 (“F2”) proteins. F1proteins and F2 proteins generally make up the water-soluble proteinportion of plant biomass. The F1 protein is an enzyme, known asribulose-1,5-bisphosphate carboxylase-oxygenase (commonly referred to asRuBisCO), whose overall molecular weight is about 550 kD (comprising alarge and small subunit with molecular weights of about 55 kDa and about13 kDa, respectively, with 8 of each subunit forming the F1 protein).RuBisCO is largely considered to be the most abundant protein in theworld, as it is present in every plant that undergoes photosynthesis.RuBisCO may comprise up to about 25% of the total protein content of aleaf and up to about 10% of the solid matter of a leaf. RuBisCO isessential to the initial step of the photosynthetic fixation of carbondioxide and functions to catalyze the carboxylation and/or oxygenationof ribulose-1,5-bisphosphate. The F2 protein is a mixture of solubleproteins of cytoplasmic and chloroplastic origin. The proteins andpeptides of the F2 protein generally have molecular weights ranging fromabout 3 kD to about 100 kD.

In certain embodiments, the protein concentrates treated according tothe methods described herein are prepared according to the methodsdescribed in U.S. Pat. No. 9,301,544 to Mua et al., which isincorporated herein by reference, the subject matter of which isoutlined briefly herein. According to the disclosure of the '544 patent,one exemplary set of processing steps that can be carried out to obtaina RuBisCO-enriched extract and/or a F2 protein-enriched extract from atobacco plant or portion thereof can include the following steps. Atobacco material is homogenized to provide a solid pulp and a liquid,protein-containing extract. The extract is clarified to remove solidstherefrom, giving a solids fraction and a clarified, protein-containingextract. The extract is then pH-adjusted and separated into a liquidcomponent and a solid, protein-containing precipitate. The precipitategenerally comprises RuBisCO as well as various additional components. Itis noted that the content of the precipitate may depend, in part, on thepH used in the previous step. For example, where the pH is between about4.5 and about 6, the precipitate may comprise more RuBisCO, whereaswhere the pH is less than about 4.5, the precipitate may additionallyinclude a significant amount of F2 proteins. The precipitate issubjected to filtration to give a protein-enriched material. Where asignificant amount of F2 fraction is contained in the liquid component,that liquid component can be treated, for example, by filtration (e.g.,through a filter or membrane on which the F2 proteins are generallyretained, while allowing certain remaining components to pass through)or by precipitation (e.g., by adjusting the pH of the liquid componentto a pH sufficient to precipitate the F2 proteins, such as less thanabout 4.5, preferably between about 3 and about 4.5).

An alternative exemplary process disclosed in the '544 patent for theproduction of a RuBisCO- and F2 protein-enriched material,RuBisCO-enriched material, and/or F2 protein-enriched material comprisesthe following steps. A tobacco material can be homogenized to provide asolid pulp and a liquid, protein-containing extract. The extract canthen be clarified to remove solids therefrom (e.g., via a pH-adjustmentstep to provide an acidic or basic clarified, protein-containingextract), giving a solids fraction and a clarified, protein-containingextract. The extract is filtered and washed to give a solid,RuBisCO-enriched material, and a permeate. In certain embodiments, thepermeate may comprise F2 proteins and the permeate can optionally beprocessed (e.g., including via filtration methods) to give an F2protein-enriched material.

In one specific embodiment, protein-enriched material 10 is obtainedaccording to the methods outlined in U.S. Pat. No. 9,301,544, asreferenced herein above, which is incorporated by reference in itsentirety. However, the disclosure is not understood to be limited toprotein-enriched material 10 obtained in this manner. Other exemplarytechniques for extracting components of tobacco are described in U.S.Pat. No. 4,144,895 to Fiore; U.S. Pat. No. 4,150,677 to Osborne, Jr. etal.; U.S. Pat. No. 4,267,847 to Reid; U.S. Pat. No. 4,289,147 to Wildmanet al.; U.S. Pat. No. 4,351,346 to Brummer et al.; U.S. Pat. No.4,359,059 to Brummer et al.; U.S. Pat. No. 4,506,682 to Muller; U.S.Pat. No. 4,589,428 to Keritsis; U.S. Pat. No. 4,605,016 to Soga et al.;U.S. Pat. No. 4,716,911 to Poulose et al.; U.S. Pat. No. 4,727,889 toNiven, Jr. et al.; U.S. Pat. No. 4,887,618 to Bernasek et al.; U.S. Pat.No. 4,941,484 to Clapp et al.; U.S. Pat. No. 4,967,771 to Fagg et al.;U.S. Pat. No. 4,986,286 to Roberts et al.; U.S. Pat. No. 5,005,593 toFagg et al.; U.S. Pat. No. 5,018,540 to Grubbs et al.; U.S. Pat. No.5,060,669 to White et al.; U.S. Pat. No. 5,065,775 to Fagg; U.S. Pat.No. 5,074,319 to White et al.; U.S. Pat. No. 5,099,862 to White et al.;U.S. Pat. No. 5,121,757 to White et al.; U.S. Pat. No. 5,131,414 toFagg; U.S. Pat. No. 5,131,415 to Munoz et al.; U.S. Pat. No. 5,148,819to Fagg; U.S. Pat. No. 5,197,494 to Kramer; U.S. Pat. No. 5,230,354 toSmith et al.; U.S. Pat. No. 5,234,008 to Fagg; U.S. Pat. No. 5,243,999to Smith; U.S. Pat. No. 5,301,694 to Raymond et al.; U.S. Pat. No.5,318,050 to Gonzalez-Parra et al.; U.S. Pat. No. 5,343,879 to Teague;U.S. Pat. No. 5,360,022 to Newton; U.S. Pat. No. 5,435,325 to Clapp etal.; U.S. Pat. No. 5,445,169 to Brinkley et al.; U.S. Pat. No. 6,131,584to Lauterbach; U.S. Pat. No. 6,284,875 to Turpen et al.; U.S. Pat. No.6,298,859 to Kierulff et al.; U.S. Pat. No. 6,772,767 to Mua et al.;U.S. Pat. No. 6,817,970 to Befit et al.; U.S. Pat. No. 6,906,172 toBratcher et al.; U.S. Pat. No. 7,034,128 to Turpen et al.; U.S. Pat. No.7,048,211 to Bratcher et al.; and U.S. Pat. No. 7,337,782 to Thompson,all of which are incorporated by reference herein. See also, theultrafiltered translucent tobacco extracts set forth in US Pat. Appl.Pub. Nos. 2013/0074855 and 2013/0074856, both to Holton, Jr., which areincorporated by reference herein.

Of course, it is to be understood that various additional processes canbe used within these exemplary methods or in addition to the steps ofthe methods in the references cited herein and the methods describedabove. For example, typical separation processes can include one or moreprocess steps such as solvent extraction (e.g., using polar solvents,organic solvents, or supercritical fluids), chromatography (e.g.,preparative liquid chromatography), clarification, distillation,filtration (e.g., ultrafiltration), recrystallization, and/orsolvent-solvent partitioning. In some embodiments, it may beadvantageous to conduct solvent extraction using a cold extractingliquid (e.g., water), particularly prior to a subsequent filtrationstep. Extractions are commonly conducted at elevated temperature;however, subjecting extracts resulting from such extractions directly tofiltration may damage the filters, due to the heat associated with theextracts and, as such, hot/warm extracts are typically cooled prior tosubsequent filtration steps. This cooling step can be avoided in someembodiments by conducting extractions at room temperature, in which casethe resulting extracts can be advantageously directly treated byfiltration. In some embodiments, the tobacco plant or portion thereofcan be pre-treated, e.g., to liberate certain compounds to make thedesired compounds available for more efficient separation. In someembodiments, multiple methods are used to obtain the desired compounds.

Other exemplary means for extraction of proteins from tobacco and otherplants include, but are not limited to, those described in U.S. Pat. No.7,337,782 to Thompson; U.S. Pat. No. 6,033,895 to Garger et al.; U.S.Pat. No. 4,941,484 to Clapp et al.; U.S. Pat. No. 4,588,691 and U.S.Pat. No. 4,400,471 to Johal; U.S. Pat. No. 4,347,324 to Kwanyuen et al.,U.S. Pat. No. 4,340,676 to Bourque; U.S. Pat. No. 4,333,871 to DeJong;U.S. Pat. No. 4,289,147 and U.S. Pat. No. 4,268,632 to Wildman et al.;U.S. Pat. Nos . 3,959,246, 3,823,128, and 3,684,520 to Bickoff et al.;US Pat. Appl. Publ. Nos. 2010/0093054 to Lo et al. and 2013/0072661 toKale; U.S. Pat. Appl. No. Int'l Appl. Publ. Nos. WO2011/078671 to Van deVelde et al. and WO2008/143914 to Lo; and EP Pat. Publ. Nos. EP 2403888to Parker et al.; EP 1691759 to Boddupalli et al.; and EP 1067946 toBrinkhaus et al., which are all incorporated by reference herein intheir entireties. Other exemplary processing methods are provided, forexample, in U.S. Pat. No. 9,220,295 to Morton et al., which isincorporated herein by reference.

A protein-enriched material 10 used as a starting point in thepurification processes discussed herein can be provided in varyingforms. In some embodiments, the protein-enriched material is provided ina moistened form. In some embodiments, the protein-enriched extract issubjected to a solvent removal process such that the extract achieves apredominantly solid form. The protein-enriched extracts may be providedin a low solvent form. By the term “low solvent form” is meant that thesolvent content including the moisture content of the material (e.g., aprotein-enriched tobacco extract) is less than about 12 percent, basedon the total weight of the material. Convenient methods for providingthe protein-enriched extract in low solvent form include spray drying,freeze drying, belt drying, flash drying, or other such methods. It isparticularly desirable to concentrate the liquid extract prior to spraydrying or freeze drying the extract. A representative spray dryingprocess is described in U.S. Pat. No. 3,398,754 to Tughan, which isincorporated herein by reference. A representative freeze drying processis described in U.S. Pat. No. 3,316,919 to Green, which is incorporatedherein by reference. Methods and conditions for providing extractedmaterials in a low solvent or solid form (e.g., as a powder) will beapparent to the skilled artisan.

The protein-enriched material 10 typically comprises some percentage ofundesirable components and/or features. For example, in someembodiments, the protein-enriched material comprises an undesirablelevel of nicotine. According to the present disclosure, aprotein-containing material (which may, in some embodiments, compriseRuBisCO and/or F2 proteins) isolated from a plant material (e.g.,according to various methods as outlined above) is further treated toprovide a protein composition (e.g., in the form of a concentrate orisolate) having a lower concentration of one or more of theseundesirable components than prior to treatment.

Generally, the methods disclosed herein involve treatment of aprotein-enriched material with peracetic acid and/or with hydrogenperoxide, as will be described in further detail herein. Treatment oftobacco and tobacco-derived materials with a range of reagents forvarious purposes is known. For example, references describing treatmentof tobacco with oxidizing agents such as ozone or hydrogen peroxideinclude, e.g., U.S. Pat. Nos. 3,612,065 and 3,943,945 to Rosen; U.S.Pat. No. 4,641,667 to Schmekel et al.; U.S. Pat. Nos. 4,366,823,4,366,824, and 4,388,933 to Rainier et al.; U.S. Pat. No. 5,713,376 toBerger; and Int. Appl. Pub. No. WO1996/31255 to Giolvas, which are allincorporated herein by reference.

In certain embodiments, the treatment process is that illustrated inFIG. 1 (Process 1). Generally, the treatment process of FIG. 1 involvesa solid material comprising protein (10), typically prepared byextracting a tobacco material to give an aqueous extract andconcentrating the extract (as described in detail herein above).Material 10 is subjected to the following steps: 12 (treating material10 with peracetic acid, giving peracetic acid-treated material 20), 22(washing peracetic acid-treated material 20 with water on a relativelylarge, e.g., micron-sized (e.g., 1.4 μm) filter to give washed material30), 32 (solubilizing washed material 30 and pH adjusting to a basic pH,e.g., about 10 or greater, to give solubilized material 40), 42(concentrating the solubilized material to give concentrated material50), and 52 (washing concentrated material 50 on a relatively small,e.g., nanometer-sized (e.g., 10 nm) filter to give purifiedprotein-containing concentrate/isolate 60).

Step 12 involves treating the protein-containing material 10 withperacetic acid to give peracetic acid-treated material 20. Optionally,prior to the addition of peracetic acid, material 10 is first pretreatedto a pH of about 3-6, e.g., around 5, which can be achieved usingvarious acids, e.g., including, but not limited to, acetic acid.

Commercially available peracetic acid is generally provided as anaqueous solution and typically contains some concentration of peroxide(H₂O₂). Thus, step 12 typically comprises treating material 10 withperacetic acid and peroxide in aqueous solution. The amount of peraceticacid employed in this step can vary, but in some embodiments is anamount of about 0.75% to about 5% peracetic acid by weight or about 1%to about 3% peracetic acid by weight, e.g., about 1% peracetic acid byweight (and about 1% to about 10% peroxide or about 3% to about 8%peroxide, e.g., about 3.5% peroxide by weight), based on the mixture ofmaterial 10, peracetic acid, peroxide, and water provided by theperacetic acid/peroxide solution. The contacting can be done at varyingtemperatures, but in some embodiments, it may enhance the treatment toemploy heating during at least a portion of step 12. For example, incertain embodiments, the mixing of protein-enriched material 10,peracetic acid and peroxide can be done at a temperature greater thanroom temperature, e.g., about 30° C. or greater, or about 35° C. orgreater, such as about 25° C. to about 42° C. or about 30° C. to about42° C. The heating temperature is generally maintained at or below about42° C. to avoid denaturing the protein. The amount of time for which theprotein-containing material 10 is maintained in contact with theperacetic acid/peroxide solution can vary, but is generally at leastabout 1 hour, at least about 2 hours, or at least about 3 hours (e.g.,about 1 to about 5 hours or about 2 to about 5 hours).

The resulting peracetic acid-treated material 20 is typically in theform of a suspension, as the protein present therein is typicallyprecipitated at low pH. Peracetic acid-treated material 20 is nextsubjected to a washing step 22. Material 20 is typically placed on arelatively large pore size filter sufficient to retain the protein(RuBisCO and/or F2 protein) on the surface of the filter. For example,material 20 can be put on a micron-sized pore filter, such as a 1.4 μmfilter (e.g., a 1.4 μm ceramic filter). In some embodiments, tangentialflow filtration is used. The washing step 22 involves contactingmaterial 20 with purified water on the filter, allowing the water toremove undesirable components, and retaining the protein on the surfaceof the filter. The water employed in the washing step advantageously hasa pH of less than 7 (i.e., somewhat acidic) to ensure that the proteindoes not solubilize/stays in a precipitated state, and does not passthrough the filter (resulting in loss of desired material). For example,the water advantageously has a pH of about 5 or less or about 4 or less(e.g., purified water with a pH of about 3.5). The water used in washingstep 22 is understood, e.g., to quench the peracetic acid remaining fromacid treatment step 12 and remove residual peracetic acid (along withpossibly other components) from the peracetic acid-treated material 20,which remains on the surface of the filter. The amount of water used towash material 20 can vary; however, a significant amount of water isgenerally employed to ensure removal of at least a majority of theperacetic acid from the sample. Similarly, the number of washingsconducted can vary, but, again, a sufficient number of washings toensure removal of at least a majority of the peracetic acid from thesample is preferred. Although not limited thereto, the washing isadvantageously conducted based on 2 to 10 times the volume of retntateand, as such, the amount of water employed in the washing (amount perwashing and number of washings) may depend upon the volume of materialbeing washed. Washing step 22 is typically conducted at roomtemperature, although this step is not limited thereto.

The resulting washed material 30 is then solubilized in step 32. Theliquid(s) used to solubilize material 30 is advantageously basic (as theproteins are understood to precipitate at low pH and solubility isenhanced as pH is increased). Consequently, step 32 typically involvescontacting material 30 with water and base. The specific base can vary,so long as it is capable of providing the desired pH and solubilizingthe protein-containing material 30. In some embodiments, a solution ofsodium hydroxide (NaOH) is used to achieve the desired pH. The pH of thesolution in which material 30 is solubilized during this step istypically about 8 or greater or about 10 or greater, e.g., about 8 toabout 16 or about 8 to about 12 (e.g., about 10.5). In some embodiments,the liquid used to solubilize material 30 further comprises a buffer.For example, in one embodiment, a buffer comprising sodium bicarbonateand potassium carbonate is used (which may include various othercomponents that do not negatively interfere with the resolubilization ofthe protein, e.g., including, but not limited to, sodium metabisulfite).Inclusion of a buffer can, in some embodiments, aid in ensuring thematerial 30 goes into solution; however, it adds salt content and thusmay require a greater extent of washing at a later step to ensureremoval of the salts.

The resulting solubilized protein-containing material, in the form ofsolubilized material 40, is then concentrated (step 42) to give aconcentrated material 50. Concentration can be done using methods knownin the art for the removal of at least a majority of the liquidassociated with solution 40. In preferred embodiments, tangential flowfiltration can be used to concentrate the material. Using tangentialflow filtration, the material 40 is put on a nanometer-sized porefilter, such as a 10 nm filter (e.g., a 10 nm ceramic filter).Advantageously, the pore size of the filter in step 42 is relativelylow; however, the pore size should not be significantly lower than 10nm, as such pore sizes will likely lead to retention of salts and otherundesired components on the surface of the filter. The retentate fromthis process then comprises concentrated material 50. In someembodiments, concentration can be done by evaporation at roomtemperature and atmospheric pressure and/or can employ elevated heat (upto about 45° C. or up to about 42° C. to avoid denaturing the protein)and/or applying vacuum). Typically, the material is concentrated so thatthe material is in substantially solid form, although some liquid maystill be associated with concentrated material 50.

The resulting material 50 is then washed 52 in a similar manner as instep 22. However, step 52 generally is conducted on a relatively smallpore size filter. For example, material 40 can be put on ananometer-sized pore filter, such as a 10 nm filter (e.g., a 10 nmceramic filter). Advantageously, the pore size of the filter in step 52is relatively low; however, the pore size should not be significantlylower than 10 nm, as such pore sizes will likely lead to retention ofsalts and other undesired components on the surface of the filter. Insome embodiments, tangential flow filtration is used. The washing step52 involves contacting material 50 with purified water on the filter,allowing the water to remove undesirable components, and retaining theprotein on the surface of the filter. The water employed in this washingstep may, in some embodiments, be purified water (e.g., having a neutralpH of ˜7). The protein is already in precipitated form at this stage ofthe process and the small pore size of the filter on which the materialis washed typically prevents significant passage of desired proteinthrough the filter.

The water used in washing step 52 is understood, e.g., to removeresidual base and salts (along with possibly other components) from theprotein-containing material 50, which remains on the surface of thefilter. The amount of water used to wash the protein-containing materialcan vary; however, a significant amount of water is generally employedto ensure removal of at least a majority of the base and salts from thesample. Similarly, the number of washings conducted can vary, but againa sufficient number of washings to ensure removal of at least a majorityof the base and salts from the sample is preferred. Although not limitedthereto, the washing is advantageously conducted based on 2 to 10 timesthe volume of retntate and, as such, the amount of water employed in thewashing (amount per washing and number of washings) may depend upon thevolume of material being washed. Washing step 52 is typically conductedat room temperature, although this step is not limited thereto. Incertain embodiments, steps 42 and 52 are combined, such thatconcentration and washing are conducted substantially together. Forexample, in certain embodiments, tangential flow filtration is employed,which can serve multiple functions, e.g., washing and concentrating theprotein-containing material.

Advantageously, material 60 (purified protein-containingconcentrate/isolate) is provided in dried or substantially dried formfollowing the washing treatment. The drying can be conducted byconcentration/evaporation and/or spray drying by methods known in theart.

Purified protein-containing material 60 generally contains a lowerconcentration of nicotine than that of a comparable material that hasnot been subjected to a process comprising steps 12, 22, 32, 42, and 52as outlined herein above. In some embodiments, the treated material(purified protein-containing concentrate/isolate 60) comprises a lowernicotine content, e.g., less than about 10 ppm or less than about 5 ppmnicotine than protein-enriched material 10. In some embodiments, thetreated material 60 exhibits a lighter color (e.g., a light cream/tancolor which is lighter in color than the material 10 subjected totreatment). In some embodiments, treated material (purifiedprotein-containing concentrate/isolate 60) has decreased ash and/ordecreased fiber with respect to the protein-containing material prior totreatment (i.e., protein-enriched material 10). For example, in someembodiments, protein-containing material 10 is at least about 40%protein, e.g., about 40% to about 60% pure protein by dry weight and thecorresponding purified protein-containing concentrate/isolate 60 is atleast about 50%, at least about 60%, or at least about 65% pure proteinby dry weight. As such, Process 1 as described herein above can, in someembodiments, lead to an increase in purity of a protein-containingmaterial of about 10% or more or about 20% or more.

In another embodiment, the treatment process is that illustrated in FIG.2 (Process 2). Generally, the treatment process of FIG. 2 involves asolid protein-enriched material (10), typically prepared by extracting atobacco material to give an aqueous extract and concentrating theextract (as described in detail herein above). Material 10 is subjectedto the following steps: 62 (adjusting the pH thereof to a basic pH,giving pH-adjusted material 70), 72 (treating the pH-adjusted materialwith hydrogen peroxide (H₂O₂), giving H₂O₂-treated material 80), 82(diluting the H₂O₂-treated material to give diluted material 90), and 92(washing the H₂O₂-treated material on a relatively small pore size,e.g., nanometer-sized (e.g., 10 nm) filter to give purifiedprotein-containing concentrate/isolate 100).

Step 62 involves treating protein-enriched material 10 with a basicsolution to give pH-adjusted material 70. In one particular embodiment,sodium hydroxide or potassium hydroxide is used as the base for thisstep, although this method step is not limited thereto and any basecapable of providing the referenced pH can be used. Typically, the baseis provided in aqueous solution and, as such, pH-adjusted material 70 isgenerally in the form of a solution. The amount of base employed in thisstep can vary, but is typically that amount needed to achieve a basic pHof the mixture, e.g., about 8 or greater, about 9 or greater, or about10 or greater (e.g., about 8 to about 11 or about 10 to about 10.5).

Although not required, the resulting pH-adjusted material 70 is thengenerally heated. The subsequent step (step 72) can be conducted at roomtemperature; however, step 72 may be more effective in some embodimentsif the pH-adjusted material is heated. Step 72 involves contacting(e.g., mixing) the pH-adjusted material with hydrogen peroxide (H₂O₂).The optional heating can be done prior to contacting material 70 withthe hydrogen peroxide and/or during the contacting. The temperature towhich material 70 is optionally heated prior to and/or during thecontacting step can vary, but in some embodiments can be a temperaturegreater than room temperature, e.g., about 30° C. or greater, or about35° C. or greater, such as about 25° C. to about 45° C. or about 30° C.to about 42° C. The heating temperature is generally maintained at orbelow about 45° C. or at or below about 42° C. to avoid denaturing theprotein.

The amount of time for which the pH-adjusted material 70 is maintainedin contact with the H₂O₂ can vary, but is generally at least about 1hour, at least about 2 hours, or at least about 3 hours (e.g., about 1to about 5 hours or about 2 to about 5 hours). The H₂O₂ with which theextract is contacted during step 72 can vary in concentration, but istypically in the form of an aqueous solution. For example, in someembodiments, commercially available H₂O₂ solutions (e.g., understood tohave various concentrations, depending on the intended application,including solutions with H₂O₂ concentration of about 3%, about 6%, orabout 34.5% by weight) is used and can be diluted to achieve the desiredoverall concentration of H₂O₂. Typically, although not limited thereto,sufficient H₂O₂ (e.g., in the form of an aqueous solution) is used toprovide an H₂O₂ concentration of at least about 1% by weight (based onH₂O₂ and water), about 2% by weight, or at least about 3% by weight(e.g., about 1% by weight to about 5% by weight, e.g., about 2% byweight to about 4% by weight).

The addition of H₂O₂ solution to pH-adjusted material 70 may result invigorous reaction. Typically, although not required, some foaming isobserved upon contact between pH-adjusted material 70 and the H₂O₂.Accordingly, in certain embodiments, the reaction is monitored andparameters are adjusted, e.g., to ensure the reaction is not overlyvigorous. For example, the pH is generally monitored to ensure a pHclose to that prior to step 70, i.e., a basic pH. The temperature isalso commonly monitored to ensure the temperature remains within theranges referenced above, particularly if heating is employed (e.g., lessthan about 45° C. or less than about 42° C., such as about 25° C. toabout 45° C. or about 30° C. to about 42° C. The mixture may optionallybe stirred during step 72 and such optional stirring is typically donegently, again to ensure the reaction is not too vigorous. The time forwhich material 70 is kept in contact with the H₂O₂ can vary andexemplary, although not limiting time periods for step 72 are about 1hour or more, about 2 hours or more, or about 3 hours or more (e.g.,about 2 to about 6 hours or about 3 to about 4 hours).

The resulting H₂O₂-treated material 80 is next diluted 82 with water togive diluted material 90. The dilution of material 80 with water servesmultiple purposes. For example, the dilution can quench remaining H₂O₂used in step 72. Dilution also helps to protect equipment (e.g.,filters) used in subsequent processing steps. The extent of dilution canvary. In some embodiments, material 80 is diluted 1:1 with water byvolume or about 1:2 with water by volume; however, the disclosure is notlimited thereto.

The resulting diluted material 90 is then washed 92, typically on arelatively small pore size filter. For example, in some embodiments,material 90 can be washed on a nanometer-sized pore filter, such as a 10nm filter (e.g., a 10 nm ceramic filter). In some embodiments, material90 can be washed on a 10 kDa membrane (e.g., spiral-wound membrane)filter. Advantageously, the pore size (or molecular weight cutoff, MWCO)of the filter in washing step 92 is relatively low; however, the poresize (or MWCO) should not be significantly lower than 10 nm (orsignificantly lower than 10 kDa) as such pore sizes/MWCOs will likelylead to retention of salts and other undesired components on the surfaceof the filter. In some embodiments, tangential flow filtration is used.The washing step 92 involves contacting material 90 with purified wateron the filter, allowing the water to remove undesirable components, andretaining the protein on the surface of the filter. The water employedin this washing step may, in some embodiments, be purified water (e.g.,having a neutral pH of ˜7). The protein is already in precipitated format this stage of the process and the small pore size of the filter onwhich the material is washed typically prevents significant passage ofdesired protein through the filter. The water used in washing step 92 isunderstood, e.g., to remove residual base and salts (along with possiblyother components) from the protein-containing material which remains onthe surface of the filter, giving purified protein-containingconcentrate/isolate 100. The amount of water used to wash theprotein-containing material can vary; however, a significant amount ofwater is generally employed to ensure removal of at least a majority ofthe base and salts from the sample. Similarly, the number of washingsconducted can vary, but again a sufficient number of washings to ensureremoval of at least a majority of the base and salts rom the sample ispreferred (e.g., 2 times to about 10 times, e.g., at least 2 times or atleast 3 times). Washing step 92 is typically conducted at roomtemperature, although this step is not limited thereto. Advantageously,purified protein-containing concentrate/isolate 100 is provided in driedor substantially dried form following the washing treatment. The dryingcan be conducted by concentration/evaporation and/or spray drying bymethods known in the art.

Purified protein-containing material 100 generally contains a lowerconcentration of nicotine than that of a comparable material that hasnot been subjected to a process comprising steps 62, 72, 82, and 92 asoutlined herein above. In some embodiments, the treated material 100comprises a lower nicotine content, e.g., about 40 ppm or less, about 30ppm or less, or about 25 ppm or less nicotine. In some embodiments, thetreated material 100 exhibits a lighter color (e.g., a light greenishcolor which is lighter in color than the material 10 subjected totreatment). In some embodiments, treated material 100 has decreased ashand/or decreased fiber with respect to the material 10 prior totreatment. For example, in some embodiments, the protein-containingmaterial 10 is at least about 40% protein, e.g., about 40% to about 60%pure protein by dry weight and the corresponding treated material 100 isat least about 50%, at least about 60%, or at least about 65% pureprotein by dry weight. As such, Method 2 as described herein above can,in some embodiments, lead to an increase in purity of aprotein-containing material of about 10% or more or about 20% or more.

Advantageously, in certain embodiments, the protein composition providedvia Method 1 or Method 2 can thus exhibit a reduced nicotine content ascompared with the protein-enriched extract. For example, in certainembodiments, the methods outlined herein can reduce the nicotine contentby at least about 20%, at least about 30%, at least about 40%, at leastabout 50%, at least about 60%, at least about 70%, at least about 80%relative to the protein-enriched extract (prior to treatment via Method1 or Method 2). For example, in some embodiments, the disclosed methodscan reduce the nicotine content by between about 5% and about 100%,e.g., between about 20% and about 100%, or between about 50% and about100%) relative to the protein-enriched extract (prior to supercriticalextraction). As referenced above, these methods can, in someembodiments, provide other benefits, e.g., with respect to taste,flavor, and/or odor. For example, in some embodiments, theprotein-enriched extract can exhibit an off-taste (e.g., a salty orotherwise negative taste), which can be significantly reduced or mutedby the disclosed methods. Similarly, in some embodiments, theprotein-enriched extract can exhibit an off-odor (e.g., an earthy odor),which can be significantly reduced or removed by the disclosed methods.Further, the protein-enriched extract can exhibit a color (e.g.,yellowish, brownish) that can be significantly reduced or removed by thedisclosed methods. The improvement in odor, taste, and/or color of thetreated material over the untreated extract can be quantified by anyknown method. For example, a sensory panel can be used to evaluate thechange in odor and or taste, whereas optical methods (e.g., lightabsorption) can be used to evaluate the change in color. Accordingly, incertain embodiments, a tasteless, colorless, and/or odorless proteinconcentrate or isolate is provided according to the methods describedherein.

The form of the protein composition provided according to the presentinvention (e.g., a RuBisCO concentrate or isolate, combined RuBisCO/F2protein concentrate or isolate, and/or F2 concentrate or isolate)obtained according to the methods of the present disclosure can vary.Typically, these materials are in solid, liquid, or semi-solid or gelforms. The resulting formulations can be used in concrete, absolute, orneat form. Solid forms of the concentrates or isolates described hereincan include spray-dried and freeze-dried forms. Liquid forms of theconcentrates or isolates described herein can include formulationscontained within aqueous or organic solvent carriers.

The methods disclosed herein may, in some embodiments, provide a proteinconcentrate or isolate comprising at least about 50%, at least about60%, at least about 70%, at least about 80%, or at least about 90%protein by dry weight. In some embodiments, the protein concentrate orisolate comprises a mixture of RuBisCO and F2 proteins. In someembodiments, the protein in the protein concentrate or isolate comprisesprimarily RuBisCO (including at least about 90%, at least about 95%, orat least about 99% RuBisCO). In some embodiments, the protein in theprotein concentrate or isolate comprises primarily F2 protein (includingat least about 90%, at least about 95%, or at least about 99% F2protein).

In some embodiments, the protein concentrate or isolate comprises atleast about 50%, at least about 60%, at least about 70%, at least about80%, or at least about 90% F1 protein by weight. In some embodiments,the present disclosure specifically provides a method for the isolationand/or purification of RuBisCO extracted from a plant of the Nicotianaspecies or a portion thereof. Accordingly, the methods disclosed hereinmay, in some embodiments, provide a RuBisCO concentrate or isolate,e.g., a material comprising at least about 50%, at least about 60%, atleast about 70%, at least about 80%, or at least about 90% RuBisCO bydry weight. In some embodiments, the protein concentrate or isolatecomprises at least about 50%, at least about 60%, at least about 70%, atleast about 80%, or at least about 90% F2 protein by dry weight.

Although in some embodiments, the protein concentrate or isolatedescribed herein can be used directly following the one or moretreatment steps, it may be desirable to thermally treat the material inorder to, for example, pasteurize the material or otherwise chemicallyalter the material. See, for example, US Pat. Pub. No. 2010/0300463 toChen et al., which is incorporated herein by reference. In someembodiments, in addition to or in place of the optional heat treatment,tobacco material can be irradiated (e.g., to ensure no microbes areassociated with the protein concentrate or isolate).

In some embodiments, the methods described herein can be used incombination with other types of treatment processes, which may, forexample, further purify or modify the concentrate or isolate. Forexample, in some embodiments, the protein-enriched composition isbrought into contact with an imprinted polymer or non-imprinted polymersuch as described, for example, in US Pat. Pub. Nos. 2007/0186940 toBhattacharyya et al; 2011/0041859 to Rees et al.; and 2011/0159160 toJonsson et al; and U.S. patent application Ser. No. 13/111,330 to Byrdet al., filed May 19, 2011, all of which are incorporated herein byreference. Treatment with a molecularly imprinted or non-imprintedpolymer can be used to remove certain components of the protein-enrichedcomposition. In some embodiments, the protein-enriched compositiondescribed herein can be subjected to conditions so as to cause compoundscontained in such material to undergo chemical transformation and/ordegradation. Exemplary chemical transformation techniques are set forthin US Pat. Appl. Pub. Nos. 2011/0174323 to Coleman, III, et al. and2011/0259353 to Coleman, III et al., which are incorporated by referenceherein. In some embodiments, other “cleanup” methods can be conductedbefore or after the disclosed methods, e.g., removal of one or more ofash, metal salts, alkaloids, particulates, heavy metals, etc. fromextracts, as disclosed in U.S. Pat. No. 9,175,052 to Gerardi et al,which is incorporated herein by reference in its entirety.

The protein concentrates and isolates (i.e., RuBisCO concentrates andisolates, combined RuBisCO/F2 protein concentrates and isolates, and/orF2 protein concentrates and isolates) provided following any one or moreof the purification treatment processes described herein canadvantageously be used in various applications. For many applications(e.g., food products, feed products, and industrial products), it may bedesirable to replace certain animal proteins with plant proteins.Additionally, in some applications, it may be desirable to replacecertain other plant proteins typically used (e.g., soy proteins and/orgenetically modified proteins). Protein concentrates and isolates suchas those provided by the methods described herein can exhibit goodnutritional properties and, in some embodiments, can be provided in aform that has a reduced content of various undesirable components (e.g.,ash, metal salts, alkaloids (e.g., nicotine), heavy metals, and otherimpurities/contaminants) and/or modified sensory characteristics (e.g.,odor, taste, and/or color), making them particularly suitable for use invarious products. Further, certain physical properties of RuBisCO renderit advantageous for use in such products, as it has excellent binding,gelling, solubility, and emulsifying behavior. In some embodiments, thetypes of treatment described herein may provide a food-gradeprotein-containing material. In certain embodiments, the proteinconcentrate isolate comprises a protein material that exceeds soyprotein in nutritional quality. In some embodiments, the proteinconcentrate or isolate may be useful for medicinal purposes.

Processed materials that are provided in accordance with the presentinvention are useful ingredients for a wide variety of commercialapplications. The materials can be used as binders, fillers orextenders, or can serve other functions or impart functional attributes,in a wide variety of industrial formulations. For example, the materialscan be used as components of various types of resins that haveindustrial applications; and additionally can be used as components ofcoatings (e.g., for inks and paints) and of adhesives (e.g., for gluesand hot melt formulations). The materials can be used as components of awide variety of cosmetic formulations (e.g., the materials can beincorporated within shampoos and skin care products). The materials canbe used as components of foods, dietary supplements and functional foods(e.g., as components of beverages, processed food products, and thelike). The materials also can be used components of animal feed. Thematerials can be used as components of pharmaceutical formulations(e.g., as components of liquids, gums, lozenges, tablets and pills thatare used for medicinal purposes). Additionally, the materials can beused as components of tobacco products; such as components of tobaccoburning products (e.g., cigarettes, cigars, pipe tobaccos, and thelike), tobacco heating smoking articles (e.g., cigarettes such as thosesold under the brand name Eclipse by R. J. Reynolds Tobacco Company),smokeless tobacco products (e.g, moist snuff, chewing tobacco, snus andso-called dissolvable tobacco products), so-called electroniccigarettes, and the like.

With regard to use in tobacco burning products, the concentrates andisolates described herein can be used in various capacities. Forexample, in certain embodiments, the concentrates and isolates can bemixed with casing materials and applied to tobacco as a casingingredient or as a top dressing, incorporated into a cigarette filter(e.g., in the filter plug, plug wrap, or tipping paper) or incorporatedinto cigarette wrapping paper, preferably on the inside surface, duringthe cigarette manufacturing process. See, for example, the descriptionand references related to tobacco isolates used in smoking articles setforth in US Pat. Pub. No. 2012/0192880 to Dube et al., which isincorporated by reference herein. Representative tobacco blends,non-tobacco components, and representative cigarettes manufacturedtherefrom are also set forth in the Dube et al. reference noted above.

Referring to FIG. 3, there is shown a smoking article 10 in the form ofa cigarette and possessing certain representative components of asmoking article that can contain the protein composition of the presentinvention. The cigarette 10 includes a generally cylindrical rod 12 of acharge or roll of smokable filler material (e.g., about 0.3 to about 1.0g of smokable filler material such as tobacco material) contained in acircumscribing wrapping material 16. The rod 12 is conventionallyreferred to as a “tobacco rod.” The ends of the tobacco rod 12 are opento expose the smokable filler material. The cigarette 10 is shown ashaving one optional band 22 (e.g., a printed coating including afilm-forming agent, such as starch, ethylcellulose, or sodium alginate)applied to the wrapping material 16, and that band circumscribes thecigarette rod in a direction transverse to the longitudinal axis of thecigarette. The band 22 can be printed on the inner surface of thewrapping material (i.e., facing the smokable filler material), or lesspreferably, on the outer surface of the wrapping material.

At one end of the tobacco rod 12 is the lighting end 18, and at themouth end 20 is positioned a filter element 26. The filter element 26positioned adjacent one end of the tobacco rod 12 such that the filterelement and tobacco rod are axially aligned in an end-to-endrelationship, preferably abutting one another. Filter element 26 mayhave a generally cylindrical shape, and the diameter thereof may beessentially equal to the diameter of the tobacco rod. The ends of thefilter element 26 permit the passage of air and smoke therethrough.

A ventilated or air diluted smoking article can be provided with anoptional air dilution means, such as a series of perforations 30, eachof which extend through the tipping material and plug wrap. The optionalperforations 30 can be made by various techniques known to those ofordinary skill in the art, such as laser perforation techniques.Alternatively, so-called off-line air dilution techniques can be used(e.g., through the use of porous paper plug wrap and pre-perforatedtipping paper). The protein composition of the invention can beincorporated within any of the components of a smoking article,including but not limited to, as a component of the tobacco charge, as acomponent of the wrapping paper (e.g., included within the paper orcoated on the interior or exterior of the paper), as an adhesive, as afilter element component, and/or within a capsule located in any regionof the smoking article.

With regard to use of the disclosed protein compositions inaerosol-generating devices that contain nicotine and/or tobacco material(or some portion or component thereof) that is not intended to becombusted during use, including so-called “e-cigarettes,” exemplarydetails are provided, for example, in US Pat. Pub. No. 2012/0192880 toDube et al., which is incorporated by reference herein. See, forexample, the various alternative smoking articles, aerosol deliverydevices and heat generating sources set forth in the background artdescribed in U.S. Pat. No. 7,726,320 to Robinson et al., U.S. patentapplication Ser. No. 13/432,406, to Griffith, Jr. et al., filed Mar. 28,2012, U.S. patent application Ser. No. 13/536,438, to Sebastian et al.,filed Jun. 28, 2012, U.S. patent application Ser. No. 13/602,871, toCollett et al., filed Sep. 4, 2012, U.S. patent application Ser. No.13/647,000, to Sears et al., filed Oct. 8, 2012, and U.S. patentapplication Ser. No. 13/840,264, to Novak, III et al., filed Mar. 15,2013, which are incorporated herein by reference.

With regard to use of the disclosed concentrates and isolates insmokeless tobacco products, exemplary products include loose moist snuff(e.g., snus); loose dry snuff; chewing tobacco; pelletized tobaccopieces; extruded or formed tobacco strips, pieces, rods, cylinders orsticks; finely divided ground powders; finely divided or milledagglomerates of powdered pieces and components; flake-like pieces;molded tobacco pieces; gums; rolls of tape-like films; readilywater-dissolvable or water-dispersible films or strips; meltablecompositions; lozenges; pastilles; or capsule-like materials possessingan outer shell and an inner region. Various types of smokeless tobaccoproducts are described or referenced in US Pat. Pub. No 2012/0152265 toDube et al., which is incorporated herein by reference. Furtheringredients can be admixed with, or otherwise incorporated within,smokeless tobacco compositions according to the invention. Exemplaryencapsulated additives are described, for example, in WO 2010/132444 toAtchley, which has been previously incorporated by reference herein. Seealso, the smokeless tobacco ingredients set forth in US Pat. Pub. Nos.2012/0055494 to Hunt et al. and 2012/0199145 to Byrd et al., which areincorporated by reference herein.

Referring to FIG. 4, a representative snus type of tobacco productcomprising a concentrate or isolate of the present invention is shown.In particular, FIG. 4 illustrates a smokeless tobacco product 40 havinga water-permeable outer pouch 42 containing a smokeless tobaccocomposition 44. Any of the components of the tobacco product cancomprise a tobacco-derived protein material as described herein (e.g.,the interior or exterior of the pouch lining or a portion of thesmokeless tobacco composition contained therein).

The amount of protein concentrate or isolate of the present inventionincorporated within a tobacco composition or tobacco product can dependon the desired function of the concentrate or isolate, the chemicalmakeup of the concentrate or isolate, and the type of tobaccocomposition to which the concentrate or isolate is added. The amount ofconcentrate or isolate added to a tobacco composition can vary, but willtypically not exceed about 50 weight percent based on the total dryweight of the tobacco composition to which the concentrate or isolate isadded. For example, the amount of concentrate or isolate added to atobacco composition may be in the range of about 0.25 to about 25 weightpercent or about 1to about 10 weight percent, based on the total dryweight of the tobacco composition.

Although the use of such protein concentrates and isolates is generallydescribed in the context of tobacco compositions, it is noted that suchformulations can be applicable in many other types of compositions,e.g., in dietary supplements as described in U.S. patent applicationSer. No. 13/830,063 to Mua et al., filed Mar. 14, 2013, which isincorporated herein by reference.

EXPERIMENTAL

Aspects of the present invention is more fully illustrated by thefollowing examples, which are set forth to illustrate certain aspects ofthe present invention and are not to be construed as limiting thereof.

Example 1 Exemplary Treatment of RuBisCO-Enriched Extract (Method 1)

Tobacco plants are harvested and the whole plants are chopped in thefield. The whole plants are extracted using a 0.75:1 weight to weightratio of buffer to tissue, using a buffer consisting of 30 mM sodiumbicarbonate, 105 mM potassium carbonate and 1% sodium metabisulfite.Fibrous material is separated from green juice, containing the protein.This green juice has a target pH of about 7.5-8.0 (and the pH is notadjusted unless far outside of this range). The green juice is chilledto less than 10° C. and the cooled green juice is passed into a decantercentrifuge at 15 LPM, 30 PSI back pressure. A solid pellet is removedfrom the liquid and the liquid is then passed to a disk stack at 8 LPM,75 PSI back pressure. Again, a solid pellet is removed therefrom and theliquid is passed to a filter press, wherein the pH of the liquid isadjusted to 6.5 with dilute HCl. The material is treated with 10 g/Ldiatomaceous earth and washed 7× volumes with extract buffer with blowdown using compressed air. The liquid passing through the filter pressis processed on a 0.1 μm ceramic filter, concentrated thereon, andwashed with a solution containing 3.0 mM NaHCO₃, 10.5 mM K₂CO₃, and 0.1%sodium metabisulfite.

The retentate from the ceramic filter is treated with 1% peracetic acidand 3.5% H₂O₂. This material is mixed at 40° C.±2° C. for 3 hours at pHabout 3.5 or lower. The treated material is processed on a 1.4 μmceramic filter. The retentate is concentrated and washed 7× withpurified water (pH 3.5). This washed retentate is resolubilized bydiluting the mixture in a 1:1 weight: weight ratio with a solutioncontaining 3.0 mM NaHCO₃, 10.5 mM K₂CO₃, and 0.1% sodium metabisulfiteand the solution is pH adjusted to 10.5 with NaOH. The resulting mixtureis processed on a 10 nm ceramic filter and the retentate is concentratedand washed 7× with purified water (pH ˜7). The washed solid is spraydried to provide a purified protein-containing material.

The material thus obtained was determined to have less than 2 ppmnicotine. In particular, the material contained about 70% amino acids bydry weight, with 120 ppm nicotine prior to treatment. After thistreatment, it contained about 80% amino acids with <1.68 ppm of nicotine(which was the limit of quantitation by analytical methods). The productalso exhibited greatly improved sensory characteristics, i.e., waslargely odorless, colorless, and tasteless.

Example 2 Exemplary Treatment of RuBisCO-Enriched Extract (Method 2)

Tobacco plants are harvested, leaves are stripped from the stalks, andthe leaves are homogenized in a disintegrator by adding water theretoand the material is then passed into a horizontal screw press for liquidextraction. The liquid protein-containing extract thus obtained is agreen juice having a pH of 8.4. This protein-containing extract isclarified first by passing the extract through a decanter.

The filtrate is concentrated on a 0.1 μm ceramic filter and washed 7×with a buffer (containing 3.0 mM NaHCO₃, 10.5 mM K₂CO₃, and 0.1% sodiummetabisulfite). Once washed, the retentate is concentrated and theretentate is pH adjusted to 10.0-10.5 by addition of a 10 N NaOHsolution. The pH adjusted retentate is heated to 40° C.±2° C. Once atthis temperature, the pH of the mixture is checked and readjusted ifnecessary to ensure a pH of 10.0-10.5. A solution of 34.5% hydrogenperoxide at 100 mL/L is added with very gentle stirring. The mixture isheld at 40° C.±2° C. and mixed gently for 3-4 hours. After this time,the mixture is diluted 2× with purified water and processed on a 10 nmceramic filter. The retentate is concentrated and washed 7× withpurified water (pH ˜7). The resulting mixture is washed 14× with USPwater and the material is reconcentrated to the initial volume. Theretentate is spray dried to provide a purified protein-containingmaterial.

The material contained about 70% amino acids by dry weight, with 120 ppmnicotine prior to treatment. After this treatment, it contained acomparable amount of amino acids with about 30 ppm of nicotine. Theproduct also exhibited greatly improved sensory characteristics, i.e.,with a significant decrease in odor and taste. The color changed from adark brown/green to a light green color.

Many modifications and other embodiments of the invention will come tomind to one skilled in the art to which this invention pertains havingthe benefit of the teachings presented in the foregoing description.Therefore, it is to be understood that the invention is not to belimited to the specific embodiments disclosed and that modifications andother embodiments are intended to be included within the scope of theappended claims.

Although specific terms are employed herein, they are used in a genericand descriptive sense only and not for purposes of limitation.

What is claimed:
 1. A method for reducing the nicotine content in aplant-derived protein-enriched material, the method comprising: a)receiving a plant-derived, protein-enriched material comprising RuBisCO,F2 fraction proteins, or a combination thereof, wherein theplant-derived, protein-enriched material further comprises undesirablenicotine; b) treating the plant-derived, protein-enriched material withperacetic acid to give a peracetic acid-treated mixture; c) washing theperacetic acid-treated mixture with water on a filter, wherein a solidtreated protein-containing material is retained on the filter; d)solubilizing the solid treated protein-containing material to give asolution and adjusting the pH of the solution to a basic pH to give abasic solution; and e) processing the basic solution on a filter toafford a retentate comprising a protein concentrate or isolate having areduced amount of nicotine as compared with the plant-derived,protein-enriched material.
 2. The method of claim 1, wherein theplant-derived, protein-enriched material comprises material from a plantof the Nicotiana species.
 3. The method of claim 1, wherein the treatingstep b) is conducted at a pH of about 3 to about
 5. 4. The method ofclaim 1, wherein the treating step b) is conducted at a temperature ofabout 25° C. to about 42° C.
 5. The method of claim 1, wherein thetreating step b) comprises treating the plant-derived, protein-enrichedmaterial with peracetic acid in the form of an aqueous solutioncomprising at least about 3% by weight peracetic acid.
 6. The method ofclaim 1, wherein step c) comprises washing the peracetic acid-treatedmixture with water on a 1.4 μm pore filter.
 7. The method of claim 1,wherein step c) comprises washing the peracetic acid-treated mixturewith water having a pH of less than about
 5. 8. The method of claim 1,wherein step d) comprises resolubilizing the solid treatedprotein-containing material in an aqueous buffer.
 9. The method of claim8, wherein the aqueous buffer comprises sodium bicarbonate and potassiumcarbonate.
 10. The method of claim 1, wherein step c) comprisesprocessing the basic solution on a 10 nm filter.
 11. The method of claim1, further comprising spray drying the protein concentrate or isolate.12. The method of claim 1, wherein the protein concentrate or isolatecomprises about 5 ppm nicotine or less.
 13. The method of claim 1,wherein the protein concentrate or isolate comprises about 80% or moreRuBisCO, F2 fraction proteins, or a combination thereof by weight.
 14. Amethod for reducing the nicotine content in a plant-derivedprotein-enriched material, the method comprising: a) receiving aplant-derived, protein-enriched material comprising RuBisCO, F2 fractionproteins, or a combination thereof, wherein the plant-derived,protein-enriched material further comprises undesirable nicotine; b)treating the plant-derived, protein-enriched material with a basicsolution to give a base-treated mixture; c) mixing the base-treatedmixture with hydrogen peroxide to give a hydrogen-peroxide-treatedmaterial; and d) processing the basic solution on a filter to afford aretentate comprising a protein concentrate or isolate having a reducedamount of nicotine as compared with the plant-derived, protein-enrichedmaterial.
 15. The method of claim 14, wherein the treating step b)comprises treating the plant-derived, protein-enriched material with thebasic solution to a pH of at least about
 10. 16. The method of claim 14,further comprising heating the base-treated mixture before, during, orboth before and during mixing step c).
 17. The method of claim 16,wherein the base-treated mixture is heated at a temperature of about 30°C. to about 42° C.
 18. The method of claim 14, wherein the processingstep d) comprises processing the basic solution on a filter with a poresize of about 10 nm or a molecular weight cutoff of about 10 kDa. 19.The method of claim 14, further comprising spray drying the proteinconcentrate or isolate.
 20. The method of claim 14, wherein the proteinconcentrate or isolate comprises about 30 ppm nicotine or less.